Redox-Cycling "Mitocans" as Effective New Developments in Anticancer Therapy

Abstract

Our study proposes a pharmacological strategy to target cancerous mitochondria via redox-cycling "mitocans" such as quinone/ascorbate (Q/A) redox-pairs, which makes cancer cells fragile and sensitive without adverse effects on normal cells and tissues. Eleven Q/A redox-pairs were tested on cultured cells and cancer-bearing mice. The following parameters were analyzed: cell proliferation/viability, mitochondrial superoxide, steady-state ATP, tissue redox-state, tumor-associated NADH oxidase (tNOX) expression, tumor growth, and survival. Q/A redox-pairs containing unprenylated quinones exhibited strong dose-dependent antiproliferative and cytotoxic effects on cancer cells, accompanied by overproduction of mitochondrial superoxide and accelerated ATP depletion. In normal cells, the same redox-pairs did not significantly affect the viability and energy homeostasis, but induced mild mitochondrial oxidative stress, which is well tolerated. Benzoquinone/ascorbate redox-pairs were more effective than naphthoquinone/ascorbate, with coenzyme Q0/ascorbate exhibiting the most pronounced anticancer effects in vitro and in vivo. Targeted anticancer effects of Q/A redox-pairs and their tolerance to normal cells and tissues are attributed to: (i) downregulation of quinone prenylation in cancer, leading to increased mitochondrial production of semiquinone and, consequently, superoxide; (ii) specific and accelerated redox-cycling of unprenylated quinones and ascorbate mainly in the impaired cancerous mitochondria due to their redox imbalance; and (iii) downregulation of tNOX.

Keywords: ascorbate; cancer; mitochondria; oxidative stress; prenylation; quinones; redox-cycling.

Figure 1

Molecular mechanism for targeting and impairing mitochondrial function by quinone and ascorbate redox-cycling in cancer cells (adapted from Bakalova et al. [9]).

Figure 2

Effects of quinone/ascorbate (Q/A) redox-pairs on cancer cell proliferation and viability—comparison with normal cells of the same origin. (A,B) The names and chemical formulas of the investigated quinones. (C) Kinetic curves of proliferation of Q/A-treated cancer cells. Untreated cells were used as control. Incubation conditions: Leukemia cells (Jurkat; 1 × 10⁶ cells/mL) were incubated with the respective Q/A combination at different concentrations within three days. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control (untreated) cells at three days incubation. The red dashed lines indicate the initial number of live cells in the samples. (D) Effects of ascorbate and respective quinones administered alone and in Q/A combination on cancer cell proliferation. Untreated cells were used as controls and cell proliferation in these samples was considered 100%. Incubation conditions: Leukemia cells (Jurkat; 1 × 10⁶ cells/mL) were incubated with the respective quinone or Q/A combination at different concentrations within 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001 versus quinone-treated cells at the respective concentration or versus untreated cells in the case of cells treated with ascorbate only (a). (E) IC50 values calculated for quinone-treated and Q/A-treated Jurkat cells within 48 h. (F) Effects of selected Q/A combinations on viability of normal lymphocytes. Incubation conditions: Normal lymphocytes (1 × 10⁶ cells/mL) were incubated with the respective Q/A combination at different concentrations within 48 h. * p < 0.05 versus untreated normal lymphocytes at the respective concentration. (G) Effects of coenzyme Q0/ascorbate (CoQ0/A) redox-pair on proliferation and viability of different cell lines: (a) colon cancer (Colon26) and normal colon epithelial (FHC); (b) breast cancer (MCF7) and normal breast epithelial (MCF10A); (c) glioblastoma (U87MG, GS9L) and normal microglial cells (EOC2). Incubation conditions: Cells (5 × 10⁵ cells/mL) were incubated with Q0/A at different concentrations within 48 h. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated cells at the respective concentration. In all charts, data are presented as means ± SD from three independent experiments with two parallel measurements per sample.

Figure 3

Effects of quinone/ascorbate (Q/A) redox-pairs on mitochondrial function of cancer and normal cells of the same origin. (A) Mitochondrial superoxide level in Q/A-treated leukemia lymphocytes (Jurkat) within 48 h analyzed by MitoSOX fluorescence. Untreated cells were used as control and MitoSOX fluorescence in these samples was considered as 100% (red dashed line). (B) Mitochondrial superoxide level in Q/A-treated normal lymphocytes within 48 h analyzed and calculated as in (A). (C) Steady-state ATP level in Q/A-treated leukemia lymphocytes (Jurkat) within 48 h analyzed by CellTiterGlo^TM luminescence. Untreated cells were used as control and ATP-based luminescence in these samples was considered as 100% (red dashed line). (D) Steady-state ATP level in Q/A-treated normal lymphocytes within 48 h analyzed and calculated as in (C). (E–G) Correlation analysis between mitochondrial superoxide, ATP level, and cell proliferation/viability in Q/A-treated leukemic lymphocytes, presented in Figure 2D and Figure 3A,C. R—correlation coefficient. (H–J) Mitochondrial superoxide level in cancer cells (Colon26, MCF7, U87MG) treated with ascorbate, CoQ0/A, K3, and AtovaQ administered alone or in combination within 48 h and analyzed by MitoSOX fluorescence. Untreated cells were used as controls and MitoSOX fluorescence in these samples was considered as 100% (red dashed line). In all charts, data are presented as means ± SD from three independent experiments with two parallel measurements per sample. All values were normalized to equal number of cells in samples. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control (untreated) cells.

Figure 4

Effects of coenzyme CoQ0/ascorbate (CoQ0/A) and menadione/ascorbate (K3/A) on cancer-bearing mice—hind paw xenografts: (A) Effect of subdermal (s.d.) injection of CoQ0/A on tumor growth in glioblastoma-grafted mice, detected within 45 days after cell (U87MG) transplantation: Control—single injection of saline solution (n = 3); 1× CoQ0/A—single injection of CoQ0/A on day 7th after cell transplantation (n = 3); 2× CoQ0/A—two injections of CoQ0/A on days 7th and 10th after cell transplantation (n = 3). CoQ0/A was injected near the tumor in a single dose 70 μg/7 mg per kg body weight (50 μL volume). Data are presented as means ± SD from 3 mice at each time point. *** p < 0.001 versus control group; ^## p < 0.01 versus 1× CoQ0/A-treated group. (B) Comparison of tumor size between control group and CoQ0/A-treated group, measured 25 days and 45 days as shown in (A). * p < 0.05, *** p < 0.001 versus control group; ^# p < 0.05, ^### p < 0.001 versus 1× CoQ0/A treated group. (C) Representative 3D magnetic resonance images of tumors in a control mouse and 2× CoQ0/A-treated mouse obtained 39 days after glioblastoma cell transplantation. (D) Effect of s.d. injection of CoQ0/A and K3/A on tumor growth in colon cancer-grafted mice, detected within 39 days after cell (Colon26) transplantation: Control—6 s.d. injections of saline solution (twice per week) (n = 6); 6× CoQ0/A—six s.d. injections of CoQ0/A (twice per week) (n = 6); 6× K3/A—six s.d. injections of K3/A (twice per week) (n = 6). CoQ0/A and K3/A were injected near the tumor in a single dose 70 μg/7 mg per kg body weight (50 μL volume), starting from day 11 after cell transplantation (red arrow). Data are presented as means ± SD from 6 mice at each time point. * p < 0.05, ** p < 0.01 versus K3/A treated group; ^# p < 0.05 versus the initial tumor size (red arrow). (E) Effect of s.d. injection of CoQ0/A and K3/A on the survival of mice described in (F). Effects of 6× CoQ0/A and 6× K3/A on tumor growth, median survival, tissue reducing capacity and tNOX expression in the tumors of colon cancer grafted mice—a comparative analysis. Data are presented as means ± SD from six mice in each group for tumor size and median survival and three mice in each group with three measurements for each specimen for TRC and tNOX assays. TRC and tNOX were analyzed on day 22 after transplantation. Samples isolated from untreated mice were used as controls. Data are expressed as % of the respective control. * p < 0.05, ** p < 0.01, *** p < 0.001 versus untreated (control) mice; ^# p < 0.05, ^### p < 0.001 versus CoQ0/A-treated mice.

Figure 5

Schematic representation of redox-cycling mechanisms of quinones with production of superoxide and hydrogen peroxide: (A) non-enzymatic ascorbate-driven one-electron redox-cycling; (B) enzyme-facilitated one-electron redox-cycling catalyzed by cytochrome P450 oxidoreductase (CYP450), thioredoxin reductase. etc.; (C) Redox-cycling mechanisms of quinone mediated by the electron-transport chain (ETC) of impaired cancerous mitochondria. Note: NAD(P)H:quinone oxidoreductase 1 (NQO1) maintains quinone in its reduced (enol) form. Thus, NQO1 restricts and even prevents non-enzymatic ascorbate-driven and enzyme-mediated one-electron redox-cycling of quinone to semiquinone and production of superoxide. One-electron oxidation of quinol in the mitochondrial ETC restores semiquinone and the subsequent generation of mitochondrial superoxide, as well as local non-enzymatic ascorbate-driven one-electron redox-cycling in the mitochondria.